Project Summary Genetics and metabolism are two defining characteristics of life. Understanding metabolism in animals is critical to unraveling the mechanistic basis of many biological processes in health and diseases, such as development, aging and cancer. For normal physiology, it is the synthesis, transformation and degradation of biomolecules (i.e., metabolic activity) that carry out the genetic blueprint of animals. For diseases, metabolic reprogramming is a hallmark for cancer (such as the Warburg effect). However, popular imaging techniques such as magnetic resonance spectroscopy, positron emission tomography, imaging mass spectroscopy and fluorescence microscopy all exhibit inherent limitations towards noninvasive high-resolution metabolic imaging. Therefore, there is no current metabolic imaging technique that can offer the desired combination of in situ probing, single-cell resolution, and volumetric imaging of three-dimensional (3D) tissues. Such technology would contribute to mechanistic understanding of normal physiology and disease progression such as tumor. The goal of this project is to develop a novel optical technology for in situ high-resolution volumetric imaging of metabolic activity in animal tissues, capturing metabolic status of every cell throughout 3D tissues. We have laid out a multi-disciplinary approach including exploration of in vivo labeling probe, novel sample treatment protocol, new microscope instrumentation construction, and multivariate computational analysis. Aim 1 is about developing heavy water (D2O) as a universal probe to be coupled with the emerging stimulated Raman scattering (SRS) microscopy, to monitor metabolic activities of tissues in a multiplex manner. Our preliminary data have demonstrated the feasibility. Advanced computational analysis and hyperspectral SRS instrumentation will be developed to achieve comprehensive metabolic profiling. Aim 2 aims to establish a volumetric imaging method to generate 3D metabolic activity maps deep into tissues. We propose to develop Raman-tailored clearing recipe that will open up volumetric SRS imaging of thick tissues and whole organs. Our preliminary data have achieved more than 10 times SRS imaging depth extension than previously possible. Aim 3 will integrate and tailor the technical development from Aim 1 and Aim 2 for imaging metabolic heterogeneity of tumor tissue. We propose to construct correlative fluorescence and SRS microscope and employ multivariate computational analysis. These development will be integrated to obtain cell-type-specific metabolic activity profiling (including different types of newly-synthesized molecules) within 3D tumor tissues, facilitating understanding of the causes, progression and refined treatment strategies of cancer. 1